It is known that antibodies can be raised to a small molecule by using a large immunogenic protein molecule as a carrier. The small molecule that derives enhanced immunogenicity by being conjugated to the carrier is called a hapten. The phenomenon of a relatively large molecule potentiating the immunogenicity of a small molecular entity to which it is attached is known in the art as the "carrier effect."
The portion of an immunogen recognized by the helper T cell (Th cell) is the T cell determinant or epitope. The portion of an immunogen that is bound by antibody is the B cell determinant or epitope. Carrier effects can be defined as immunity to one determinant, the "helper" or T (T.sub.h) cell determinant, of a multideterminant immunogen enhancing the immune response to another determinant, the B cell determinant.
It is now well established that most immunogens require T cell help to induce B cells to produce antibodies. Thus, T.sub.h cells, by recognizing helper determinants on the immunogen help B cells to make antibody against the immunogen.
The antigenic determinants recognized by T helper cells (T.sub.h) and B cells must be associated to form a single molecular entity, but they do not have to be covalently linked. See, Russel et al., Nature, 280:147 (1979), Lamb et al., J. Immunol., 129:1465 (1982), Scherle et al., J. Exp. Med., 164:1114 (1986) and Lake et al., Cold Spring Harbor Symp. Quant. Biol., 41:589 (1976). Some immunogens do not require T cell help to induce antibody formation, these are T-independent antigens.
A pathogen-related protein can be immunologically mimicked by the production of a synthetic polypeptide whose sequence corresponds to that of a determinant of the pathogen. Such polypeptide immunogens are reported by Sutcliffe et al., Nature, 287:801 (1980), and Lerner et al., Proc. Natl. Acad. Sci. USA, 78:3403 (1981).
Gerin et al., Proc. Natl. Acad. Sci. USA, 80:2365 (1983), showed limited protection of chimpanzees from hepatitis B virus upon immunization with carrier-bound synthetic polypeptides having amino acid residue sequences that correspond to the sequence of a determinant portion of HBsAg; in particular, residues 110-137 of the "S" (surface) region. However, the carrier protein used in those studies was keyhole limpet hemocyanin (KLH), a T cell-dependent carrier that is not fit for use in medical applications to humans because it is a source of irritation that leads to severe inflammation.
T cell-stimulating carrier proteins capable of enhancing the immunogenicity of haptens that do not produce unacceptable side effects in human subjects are often immunogenic natural proteins. For example, tetanus toxoid (TT) has been frequently used when a carrier suitable for human administration was needed. However, the use of tetanus toxoid as a carrier was restricted due to problems with dosage limitations and risk of sensitization to the toxoid itself. In addition, an epitope-specific suppression of response to the carried hapten can occur in individuals already immunized against tetanus.
The hepatitis B surface protein has been proposed as a carrier for heterologous epitopes. Delpeyroux et al., Science, 233:472-475 (1986), reported the use of the HBV surface protein (S protein) as a carrier for a poliovirus polypeptide hapten. Those investigators constructed a recombinant deoxyribonucleic acid (DNA) protein expression vehicle that produces a fusion protein, designated HBsPolioAg, capable of forming particles closely resembling authentic 22-nanometer HBsAg particles. HBsPolioAg consists of HBV S protein having an 11 amino acid residue sequence insert corresponding to amino acids 93-103 of capsid protein VPI of poliovirus type 1 (Mahoney strain).
Hepadnavirus nucleocapsid proteins have been used as hapten carriers. Heterologous immunogenic peptide sequences inserted internally in the hepatitis B core, expressed as fusion particles, elicited very high immune responses in immunized animals in the absence of adjuvants. B. E. Clarke et al. Vaccines 91:313-318 (1991); F. Schodel et al. J. Virol. 66(1):106-114 (1992). U.S. Pat. Nos. 4,818,527, 4,882,145, and 5,143,726, the disclosures of which are incorporated herein by reference, describe the use of the carrier effect with hepatitis B virus nucleocapsid protein to enhance the immunogenicity of an operatively linked polypeptide hapten. Those patents describe the linking of a polypeptide hapten to hepatitis B virus nucleocapsid protein through an amino acid residue side chain that occurs naturally in the hepatitis B nucleocapsid protein sequence.
Hepadnavirus nucleocapsid proteins are fairly well studied. SEQ ID NOs:1 and 2 are the DNA and amino acid sequences of the human hepatitis B core protein (HBc), subtype ayw, as described in U.S. Pat. Nos. 4,818,527, 4,882,145, and 5,143,726. Other hepadnavirus nucleocapsid protein sequences are also known in the art, see e.g. SEQ ID NOs: 3-13.
There are reasons to select hepadnavirus nucleocapsid proteins as a carrier over other carriers used in the art, such as keyhole limpet hemocyanin (KLH), BCG, tetanus toxoid and diphtheria toxoid. KLH, BCG, tetanus toxoid and diphtheria toxoid are non-particulate, whereas hepadnavirus nucleocapsid proteins tend to aggregate into "particles". HBc particles tend to have a higher immunogenicity than hepatitis B surface antigen (HBsAg) particles. D. R. Milich et al., Science, 234:1398-1401 (1986). HBc is both a T cell-independent and a T cell-dependent immunogen. Id. HBc is one of the most immunogenic proteins known. Almost all hepatitis B-infected people develop a powerful immune response to core. J. H. Hoofnagle, Semin. Liver Dis., 1(1):7-14 (1981). HBc can provide universal responsiveness, irrespective of genetic background. Id. HBc directly activates T cells. HBc elicits strong T.sub.h cell responses. HBc is efficiently processed and presented by antigen-presenting cells. Due to the inherently high immunogenicity of HBc, complex adjuvants are typically not required, for example, the common and inexpensive alum is sufficient.
The family hepadnaviridae is a family of enveloped animal viruses with a core of DNA that cause hepatitis B in humans. The hepadnaviridae are not responsible for human hepatitis A (a single-stranded RNA enterovirus), human hepatitis C (Flaviridae family of single stranded RNA virus), or human hepatitis D (a closed circular negative-sense RNA satellite virus, "delta virus", that requires hepatitis B virus for replication). The hepadnaviridae family includes hepatitis viruses of other species, e.g. woodchuck, duck, ground squirrel, and heron, in addition to human and simian hepatitis B. Hepatitis B (HB) used hereinafter refers to the family hepadnaviridae, unless the discussion is referring to a specific example.
Hepatitis B core protein monomers self-assemble into stable aggregates known as hepatitis B core protein particles (HBc particles). For example, human HBc particles are 27 nanometers (nm) in diameter. Conway et al., Nature, 386:91-94 (1997), describe the structure of human HBc particles at 9 .ANG.ngstrom resolution, as determined from cryo-electron micrographs. Bottcher et al., Nature, 386:88-91 (1997), describe the polypeptide fold for the human HBc monomers, and provide an approximate numbering scheme for the amino acid residues at which alpha helical regions and their linking loop regions form. Bottcher et al. propose a loop from about residues 78 to 82 of the hepatitis B core protein.
Using synthetic peptides and monoclonoal antibodies, the immunodominant loop region of HBc was mapped to about amino acid residues 75 to 83. G. Colucci et al., J. Immunol., 141:4376-4380 (1988). Two immunodominant linear epitopes were reported by other workers at amino acid residues 75 to 85 and 130 to 140. Salfeld et al. J. Virol. 63:798 (1989).
Insertion mutants of the hepatitis B core protein still are able to form core particles when foreign epitopes are cloned into the immunodominant loop region of HBc. P. Pumpens et al., Intervirology, 38:63-74 (1995). The HBc fusion proteins form particles in prokaryotic and eukaryotic expression systems. Id.
The ability to use a protein as a carrier for a pendently-linked hapten depends upon several factors that have been studied with respect to HBc. Chemically-reactive amino acid side chains, such as lysine (K), aspartic acid (D), glutamic acid (E), and reduced cysteine residues (C), provide functional groups that can be useful for modifying polypeptides.
The hepatitis B core protein sequence has several chemically-reactive amino acid side chains in the native sequence. Core has three primary amino groups, one at the amino terminus, and two lysine residues (K5 and K96), along with four cysteine residues (C48, C61, C107 and C183). There are several carboxylic acid groups, D (2, 4, 22, 29, 32, 78, 83) and E (8, 14, 40, 43, 46, 64, 77, 113, 117, 145, 179) and the carboxy terminus.
However, the native, unmodified hepatitis B core protein particle does not exhibit appreciable chemical reactivity of the amino acid side chains in the native sequence. The chemical reactivity of an amino acid side chain in a protein depends upon the nature of the amino acid side chain, and its environment in the folded protein.
As is discussed in detail hereinafter, it has now been found that the problem of low reactivity of the amino acid side chains in native hepatitis B nucleocapsid protein can be overcome by inserting a chemically-reactive amino acid side chain into the HBc protein sequence. A strategically modified hepadnavirus core protein particle of the present invention exhibits substantially enhanced reactivity toward derivatization of HBc particles with chemically linked haptens, and provides enhanced immunogenicity to those linked haptens.
These modified HBc proteins and their pendently-linked hapten conjugate derivatives are discussed in the disclosure that follows.